Structural determination of carbohydrates is mostly performed by liquid-state NMR, and it is a demanding task because the NMR signals of these biomolecules explore a rather narrow range of chemical shifts, with the result that the resonances of each monosaccharide unit heavily overlap with those of others, thus muddling their punctual identification. However, the full attribution of the NMR chemical shifts brings great advantages: it discloses the nature of the constituents, the way they are interconnected, in some cases their absolute configuration, and it paves the way to other and more sophisticated analyses. The purpose of this review is to provide a practical guide into this challenging subject. It will drive through the strategy used to assign the NMR data, pinpointing the core information disclosed from each NMR experiment, and suggesting useful tricks for their interpretation, along with other resources pivotal during the study of these biomolecules.

Nonulosonic acids are found in the surface polysaccharides of many bacterial species and are often implicated in pathogenesis. Here, the structure of a novel 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acid recovered from the capsular polysaccharide of a multiply antibiotic resistant Acinetobacter baumannii isolate was determined. The isolate carries a sugar synthesis module that differs by only a single gene from the module for the synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulosonic acid or 5,7-di-N-acetylacinetaminic acid, recently discovered in the capsule of another A. baumannii isolate. The new monosaccharide is the C8-epimer of acinetaminic acid (8eAci; 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-L-altro-non-2-ulosonic acid) and the C7-epimer of legionaminic acid. This monosaccharide had not previously been detected in a biological sample but had been synthesized chemically.

Fusobacterium nucleatum is a common member of the oral microbiota. However, this symbiont has been found to play an active role in disease development. As a Gram‐negative bacterium, F. nucleatum has a protective outer membrane layer whose external leaflet is mainly composed of lipopolysaccharides (LPSs). LPSs play a crucial role in the interaction between bacteria and the host immune system. Here, we characterised the structure of the O‐antigen and lipid A from F. nucleatum ssp. animalis ATCC 51191 by using a combination of GC‐MS, MALDI and NMR techniques. The results revealed a novel repeat of the O‐antigen structure of the LPS, [→4)‐β‐d‐GlcpNAcA‐(1→4)‐β‐d‐GlcpNAc3NAlaA‐(1→3)‐α‐d‐FucpNAc4NR‐(1→], (R=acetylated 60 %), and a bis‐phosphorylated hexa‐acylated lipid A moiety. Taken together these data showed that F. nucleatum ATCC 51191 has a distinct LPS which might differentially influence recognition by immune cells.

The giant virus Mimivirus encodes an autonomous glycosylation system that is thought to be responsible for the formation of complex and unusual glycans composing the fibers surrounding its icosahedral capsid, including the dideoxyhexose viosamine. Previous studies have identified a gene cluster in the virus genome, encoding enzymes involved in nucleotide-sugar production and glycan formation, but the functional characterization of these enzymes and the full identification of the glycans found in viral fibers remain incomplete. Because viosamine is typically found in acylated forms, we suspected that one of the genes might encode an acyltransferase, providing directions to our functional annotations. Bioinformatic analyses indicated that the L142 protein contains an N-terminal acyltransferase domain and a predicted C-terminal glycosyltransferase. Sequence analysis of the structural model of the L142 N-terminal domain indicated significant homology with some characterized sugar acetyltransferases that modify the C-4 amino group in the bacillosamine or perosamine biosynthetic pathways. Using mass spectrometry and NMR analyses, we confirmed that the L142 N-terminal domain is a sugar acetyltransferase, catalyzing the transfer of an acetyl moiety from acetyl-CoA to the C-4 amino group of UDP-d-viosamine. The presence of acetylated viosamine in vivo has also been confirmed on the glycosylated viral fibers, using GC-MS and NMR. This study represents the first report of a virally encoded sugar acetyltransferase.

The general perception of viruses is that they are small in terms of size and genome, and that they hijack the host machinery to glycosylate their capsid. Giant viruses subvert all these concepts: their particles are not small, and their genome is more complex than that of some bacteria. Regarding glycosylation, this concept has been already challenged by the finding that Chloroviruses have an autonomous glycosylation machinery that produces oligosaccharides similar in size to those of small viruses (6–12 units), albeit different in structure compared to the viral counterparts. We report herein that Mimivirus possesses a glycocalyx made of two different polysaccharides, now challenging the concept that all viruses coat their capsids with oligosaccharides of discrete size. This discovery contradicts the paradigm that such macromolecules are absent in viruses, blurring the boundaries between giant viruses and the cellular world and opening new avenues in the field of viral glycobiology.